Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp. 536-540, March 1971 Kinetics of Insulin Release from the Perfused Rat Pancreas Caused by , Glucosamine, and RUDIGER LANDGRAF, JANINA KOTLER-BRAJTBURG, AND FRANZ M. MATSCHINSKY Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by Oliver H. Lowry, December 18, 1970

ABSTRACT Under appropriate conditions, not only added to provide a colloid osmotic pressure similar to that of glucose but also glucosamine and galactose can serve as serum. 1 hr prior to perfusion, the medium was warmed to potent stimulants for insulin release from the isolated, resulting pH probably, 350C and equilibrated with Or-CO2 95:5. The perfused rat pancreas. Since galactose and, less than 0.1 unit during one passage glucosamine are not metabolized in the islets, and since was 7.4. The pH changed these three compounds have in all likelihood common through the organ complex. The perfusion pressure varied be- sites of action, it is postulated that a glucoreceptor ofbroad tween 40 and 80 mm of Hg. The average flow rate was 7.0 specificity is involved in the mechanism of insulin release, ml/min and was constant throughout the experiment, which and that metabolism of glucose is not an essential part of usually lasted 1 hr. the releasing action of this . After the pancreas was perfused for 10 min with the same An important but deceptive question about the islets of Lang- medium as used during the control periods of each experiment, erhans is how glucose stimulates insulin release. The question the effluent was sampled. Usually three samples were taken as is important because it concerns a crucial aspect of normal controls. At intervals, indicated in the figures, the perfusate function and of diabetes; it is deceptive because any specific was quickly changed by switching from one circulation releasing functions of glucose can be easily obscured by the medium to another and sampling was continued. The dead the con- more general role of glucose as a fuel. A promising approach to space of the combined arterial and venous side of this difficulty is to compare the effects of glucose with its necting tubes and cannulas was about 400 Al. This dead space, epimers (e.g., galactose), derivatives (e.g., glucosamine), and together with the extracellular water space of the whole prep- other closely related compounds, (e.g., mannoheptulose) and, aration, determined the resolving time for stimulation and change in this way, to attempt to distinguish release and fuel func- sampling. Transition between conditions upon medium tions. The perfused pancreas, although technically somewhat took 45-60 see as judged by glucose levels in the perfusate. demanding (1), seemed an ideal system for such investigations. Immediateiy after sampling, the tubes containing aliquots were In this paper we describe the peculiar kinetics of insulin (about 0.5 ml) of perfusate were placed on dry ice. They - secretion observed when the perfused pancreas of the rat was stored at 80'C until used. Insulin concentration in the per- stimulated by glucose, galactose, or glucosamine alone, by fusate was measured in duplicate by the immunoassay of combinations of subthreshold levels of glucose with galactose Hales and Randle (3), using pork insulin as standard. or glucosamine, and also by combinations of stimulatory RESULTS amounts of any of the three with theophylline. The effects of glucose (Figs. 1 and 2) MATERIALS AND METHODS Insulin release induced by 20 mM glucose was multiphasic. All reagents were analytical grade. (clinical grade) The initial phase began after a short delay (45 see) and and D-galactose were purchased from Mann Research Lab- reached its maximum within 2.5-3 min. After a transient de- oratories. D-glucosamine was obtained from Sigma Chemical cline at a low point at 7 min, a second, gradually rising, re- Co. D-glucOse was purchased from Mallinckrodt Chemicals sponse occurred; this response disappeared upon cessation of and theophylline from Matheson, Coleman and Bell. Dextran the glucose stimulus. Prestimulatory insulin levels were was dialyzed against deionized water to remove traces of con- reached within 4 min after cessation of glucose perfusion. taminant glucose. Galactose was free of glucose as judged by This biphasic response is well known from other studies with enzymatic fluorometric assay (2), but glucosamine contained perfused rat pancreas (4) and from in vivo studies with dog up to 1% glucose. (5) and man (6). Male Sprague-Dawley rats, 300-500 g, fed ad libitum with The responsiveness of islets of Langerhans to stimulation Purina chow and water, served as pancreas donors. with glucose was preserved (Fig. 1, control) when the pan- The pancreas was isolated and perfused using the procedure creas was perfused for an additional 40 min with substrate- described by Grodsky et al. (1) with minor modifications. The free solution. (We assume that pancreatic islets are unable to animals were fasted overnight, treated with atropine (0.1 utilize the high molecular weight dextran.) The glucose-pro- mg/kg) intraperitoneal, and anesthetized with pentobarbital voked response at this late stage was somewhat delayed, with (45 mg/kg, intraperitoneal). The pancreas was removed to- insulin first appearing in the perfusate 2-3 min after addition gether with the spleen, the stomach, and the proximal part of of the sugar. Nevertheless, the typical biphasic pattern was the duodenum. The celiac axis and the portal vein were can- preserved. nulated and the organ complex was placed in an incubator at When substimulatory concentrations of glucose (3 mM) constant temperature (350C). Perfusate was not recycled. were present in the perfusate during the period before per- The perfusion fluid had the following composition: NaCl, fusion with 20 mM glucose, a more rapid response to high 120 mM; KCl, 4.7 mM; MgSO4, 0.8 mM; CaCl2, 2.5 mM; glucose was observed (Fig. 2). When, after 40 min of stimula- to 3 KH2PO4, 1.2 mM; and NaHCO3, 25 mM. Dextran (8%) was tion, the glucose concentration was decreased again mM, 536 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971). Glucoreceptor and Insulin Release 537

20 25 30 35 40 DURATION OF PERFUSION, MIN FIG. 1. Release of insulin due to glucose, theophylline, and combinations of these two agents. During the control periods (-3 to 0 and 40 to 65 min) the perfusate was substrate free, except for the control experiment in which the perfusate was substrate free for 40 min, at which time 20 mM glucose was introduced. The concentrations of glucose and theophylline were 20 and 10 mM, respectively. Each re- leasing-profile represents one of three experiments performed under the same conditions. All gave comparable data. This approach was followed throughout the study, except in the case of the single experiment presented in Fig. 3.

DURATION OF PERFUSION, MIN FIG. 2. Release of insulin due to glucose. The perfusate contained 3 mM glucose during the control periods (-3 to 0 and 40 to 54 mm in Expt. A, and -3 to 40 min in Expt. B). High glucose (20 mM) was present from 0 to 40 min and from 40 to 54 min in experiments A and B, respectively. Each curve represents the results of a typical experiment, which was repeated 3 times with comparable outcome.

a small burst (+50%) of insulin release occurred, which The effects of glucosamine (Figs. 3 and 4) lasted about 1 min. When the switch from low to high glucose The most notable effect occurred when theophylline was in- was delayed (until 40 min later than the experiment above) fused prior to and during the exposure to glucosamine (Fig. 3). release occurred swiftly. The maximum release was reached A brief initial, and a delayed secondary, phase of secretion within 1 min, rather than at 5 min as seen in the comparable were produced, similar to the profile after glucose perfusion experiment without a low concentration of glucose in the pre- (compare Figs. 1 and 3). A pronounced "off-response" was ob- liminary perfusion. served when glucosamine was removed without discontinuing Theophylline by itself caused hormone release (Fig. 1) with theophylline infusion (Fig. 3). This "off-effect" was even a slow onset. The insulin concentrations in the perfusate more marked when both theophylline and the sugar were dis- reached a plateau within 4 min. After cessation of theophyl- continued simultaneously (Fig. 4). When glucosamine was in- line perfusion, the insulin output declined slowly to above- fused alone, or in combination with 3 mM glucose, the only prestimulatory concentrations. Theophylline greatly poten- consistent result was a small "off-response" occurring after tiated the effect of glucose, but the pattern of release was not removal of glucosamine from the perfusate (Fig. 4). altered qualitatively. The stimulatory action of methyl xan- Coore and Randle, using pieces of rabbit pancreas, demon- thines on insulin release has been amply documented (7-9). strated that glucosamine exhibits stimulatory or inhibitory Downloaded by guest on September 29, 2021 538 Physiology: Landgraf et al. Proc. Nat. Acad. Sci. USA 68 (1971)

160 }0 %TrHEOPHYLLINE THEOPHYLLINE+GLUCOSAMINE HEOPMLLINE CONTROL 0 200 c

t 120 z 81 200 D^

) 80 010

z- '2' 40

0 41

-3 0 5 10 15 20 25 30 35 40 45 50 55 60 DURATION OF PERFUSION, MIN FIG. 3. Glucosamine as a stimulus for insulin release after priming with theophylline. The concentration of glucosamine (infused from 17 to 42 min) was 20 mM. Theophylline (10 mM) was present from 0 to 52 min. Theophylline treatment was preceded and followed by perfusion with substrate-free medium. The data are the results of a single experiment of this kind.

2800 2800

2000 ~~~~~~~~~~~~~~~~~~~~~~2000 GLUCOSAMINE + C THEOPHYLLLINE 1200 1600

92800 ; GLUCOSAMINEGLUOSAINEGLUCOSAMINE( + 4 400-40

0- -3 0 10 15 20 25 30 35 40 45 50 55 60 65 DURATION OF PERFUSION,MIN FIG. 4. Release of insulin due to glucosamine aLd combinations of the with glucose or theophylline. In the experiment designated "glucosamine + glucose", 3 mM glucose was present from the beginning (-3 min) to the end of the perfusion. In the other two experiments, the perfusate had no additions before to. The concentrations of glucosamine a d theophylline were 20 and 10 mM, respectively. At 40 min, perfusion was started with medium free of sugar or theophylline. The results of typical individual experiments are recorded (see also legend to Fig. 1).

action depending on whether low or high concentrations of from ,8-cells, since Diazoxide and cyanide were able to block glucose are added to the incubation fluid (10). Lambert et al. this phase of release (not shown). When subthreshold concen- observed that glucosamine is a potent stimulator of embryonic trations of glucose were present from the beginning to the end pancreas explants in the presence of caffeine (9). The sudden of the experiment, the initial response to galactose was equally burst in insulin output seen in the present study after re- great and occurred without lag, yet the second phase and the moval of high concentrations of glucosamine from the per- prolonged secretion persisting after removal of the sugar from fusate might be regarded as a sign of release from inhibition. the perfusate, seen with galactose alone, were missing. Detailed dose-response studies with this sugar promise to pro- When theophylline was present, galactose induced a pat- vide more insight into the mechanism(s) involved. tern of hormone secretion that resembled the pattern pro- duced by glucose: a pronounced early peak followed by a slow The effects of galactose (Fig. 5) second phase of release, and finally, an exponential decay of Galactose caused insulin secretion under all conditions tested. insulin output after removal of the sugar. The kinetics of the endocrine responses differed characteristi- The relative potency for insulin release of galactose, com- cally, depending upon the experimental situation. When 20 pared to glucose, ranged from 10-70% depending upon the mM galactose was infused alone, the onset of the small initial conditions. phase was delayed. There followed a transient shutdown of The duration of the "off-effects" varied greatly, depending secretion, then a second secretory phase which became mani- on the conditions. The effect lasted 1 min after theophylline fest about 15 min later. Finally, when galactose was removed plus glucose, 5 min after theophylline plus glucosamine, and from the perfusate, insulin release increased progressively more than 25 min after galactose. One plausible explanation throughout the remaining period of 25 min, reaching very for this phenomenon might be that all three stimulants are in- high concentrations. This increased insulin output seems to be hibitory at high concentrations and that the duration of the due to active secretion rather than leaking of the hormone off-effect is inversely proportional to the rate of efflux of the Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Glucoreceptor and Insulin Release 539

1200 - z

:1 Li 800 lo

0A

400 -4_) Cz z

DURATION OF PERFUSION, MIN FIG. 5. Release of insulin due to galactose alone and in the presence of theophylline or glucose. In the experiment labeled "galactose + glucose", 3 mM glucose was present from the beginning to the end of the perfusion. In the other two experiments, the perfusion medium had no additions before to and after 40 min. The concentration of galactose was 20 mM and that of theophylline 10 mM. As in Figs. 1, 2, and 4, each curve represents the results of a typical experiment, which was repeated at least 3 times with comparable outcome. Secretory Complex IV

Insulin * /I,,-

G - 6 -P ----. n tormadlooes A DR -'- ATrP _7 ose0 - -- Igu9 cose :P-rG-P6-P

TPN - - TPNH A T D _ 3'5'AMP, Adenyl Cyclase

35AMP

- I II III Glucose Carrier and/or Glucose Activation In termediary Receptor Complex Metabolism FIG. 6. Outline of glucoreceptor theory. For explanation, see text of the Discussion section.

respective sugar from the islet cells. It is known that glucose serum albumin, used by most investigators as a colloid os- moves extremely quickly across the cell membrane (11). motic agent, might change the responsiveness of the jB-cells Whether, as this hypothesis demands, glucosamine and to galactose. galactose enter and leave the cell relatively slowly remains to be tested. DISCUSSION In most other in vitro studies (1, 2, 10, 12, 13) galactose has The data presented here make it difficult to accept in an un- consistently failed to release insulin. However, Lambert et al. modified form the hypothesis that attributes glucose-stim- showed stimulation of insulin release by galactose with ex- ulated insulin release to metabolism of the sugar (1, 12, 14). plants of fetal pancreas (9) and noted that the addition of If we accept the amply supported finding that islets are un- caffeine was an absolute requirement for this effect. It is able to metabolize galactose (15, 16), and if we assume that possible that the effect of galactose has been obscured pre- galactose and glucose have common site(s) of action in the j#- viously because of the use of short periods of stimulation or cell, we must postulate a mechanism that involves direct the use of subthreshold concentrations of glucose which, as stimulation of the #-cells by the intact sugars. This does not shown in these experiments, blocks all but the initial brief re- imply that metabolism of the stimulating sugar is without sponse. It is also possible that the availability of excess fuel influence on the response. In fact, differences in the character from the high concentration of free fatty acids present in and magnitude of the responses may-at least in part-turn Downloaded by guest on September 29, 2021 540 Physiology: Landgraf et al. Proc. Nat. Acad. Sci. USA 68 (1971)

out to be due to differences in the metabolism of the stimulants These studies were supported by USPHS research grants AM by the islet cells. 15092 and NB 08000, and by Career Development Award GM One recent finding published by Renold and his coworkers 42374. deserves consideration in this context (17). These investiga- 1. Grodsky, G. M., A. Batts, L. L. Bennett, C. Vcella, N. B. tors observed a paradoxical enhancement of tolbutamide Williams, and D. F. Smith, Amer. J. Physiol., 205, 638 (1963). action by 2-deoxy-D-glucose and D-mannoneptulose, two po- 2. Lowry, 0. H., J. V. Passonneau, F. X. Hasselberger, and tent inhibitors of glucose-stimulated insulin release. This D. Schulz, J. Biol. Chem., 239, 18 (1964). might be explained by postulating a mechanism that involves 3. Hales, C. N., and P. J. Randle, Biochem. J., 88, 137 a tolbutamide receptor in close proximity to a sugar (1963). receptor 4. Curry, D. L., L. L. Bennett, and G. M. Grodsky, Endo- of broad specificity. If this were the case, one might expect crinology, 83, 572 (1968). that glucose would also potentiate tolbutamide action, and in 5. Kanazawa, Y., T. Kuzuya, T. Ide, and K. Kosaka, Amer. fact there are reports in the literature that may be inter- J. Physiol., 211, 442 (1966). preted as supporting this idea (12, 18). It has also been ob- 6. Cerasi, E., and R. Luft, Acta Endocrin., 55, 278 (1967). 7. Sussman, K. E., G. D. Vaughan, and M. R. Stjernholm, served (A. Lambert, personal communication) that 3-0- in Diabetes, ed. J. Ostman and R. D. G. Milner (Excerpta Medica methylglucose potentiates the insulin release due to caffeine, a Foundation, Amsterdam, 1969), p. 123. result best explained if a site independent of metabolism was 8. Turtle, J. R., G. K. Littleton, and D. M. Kipnis, Nature, involved in the release mechanism. (London), 213, 727 (1967). Another line of evidence, which speaks against the 9. Lambert, A. E., A. Junod, W. Stauffacher, B. Jeanrenaud, primary and A. E. Renold, Biochim. Biophys. Acta, 184, 529 (1969). role of glucose metabolites as triggers of release, comes from 10. Coore, H. G., and P. J. Randle, Biochem. J., 93, 66 recent biochemical studies of the islets of Langerhans of the (1964). rat (19, 20). The concentrations of most of the early metab- 11. Matschinsky, F. M., and J. Ellerman, J. Biol. Chem., 243, olites and of cofactors of glucose metabolism were unchanged 2730 (1968). 12. Malaisse, W., Etude de la S&crgtion Insulinique In Vitro during the first 5 min of glucose infusion, in spite of abundant (Editions Arscia, Brussels, 1969). insulin release, but the metabolite pattern of the islets 13. Lacy, P. E., D. A. Young, and C. F. Fink, Endocrinology, changed markedly after prolonged glucose infusion. Accord- 83, 1155 (1968). ingly, intermediates such as glucose-6-phosphate (11, 14) and 14. Randle, P. J., S. J. H. Ashcroft, and J. R. Gill, in Carbo- 6-phosphogluconate (21), which have been implicated as sig- hydrate Metabolism and Its Disorders, ed. F. Dickens, P. J. Randle, and W. J. Whelan (Academic Press, 1968), p. 427. nals, hardly qualify for such a role. 15. Jarret, R. J., and H. Keen, Metabolism, 17, 155 (1968). Therefore, we have adopted an alternative working hy- 16. Hellerstrom, C., Endocrinology, 81, 105 (1967). pothesis (Fig. 6) that could explain the major actions of glucose 17. Renold, A. E., Y. Kanazawa, A. Lambert, L. Orci, J. in #-cells of the rat (19, 20). Burr, L. Balant, D. W. Beaven, G. M. Grodsky, W. Stauffacher, B. Jeanreneaud, and C. Roviller, New Eng. J. Med., 282, 173 Four facets of glucose action are represented in the sketch: (1970). I, the glucose receptor complex; II, glucose activation; III, 18. Lambert, A., D. Vecchio, A. Gonet, B. Jeanreneaud, and intermediary metabolism; and IV, the secretory complex. A. Renold, in Tolbutamide after Ten Years, ed. W. J. H. Butter- According to the hypothesis, glucose itself acts as a stimulus field and W. van Westering (Excerpta Medica Foundation, for insulin release at the receptor (I), presumably located in Amsterdam, 1967), p. 61. 19. Matschinsky, F. M., J. Ellerman, J. Kotler-Brajtburg, the cell membrane and tentatively called glucoreceptor. This J. Krzanowski, R. Landgraf, and R. Fertel, J. Biol. Chem., in site is either part of a carrier system or is an independent site. press. It is accessible from the outside or from the inside of the cell. 20. Matschinsky, F. M., Advances of Clinical Biochemistry, The primary event, which is set off by glucose per se, is an in- ed. U. Dubach and U. Schmidt (Hans Huber Publishers, Bern, crease of ion and Na+ and Switzerland), in press, vol. 3. conductivity (Ca++, perhaps K+) 21. Montague, W., and K. W. Taylor, Biochem. J., 109, 333 with insulin secretion as the consequence. The essential role (1968). of Ca++ for excitation-secretion coupling in general, and for 22. Malaisse, W. J., and F. Malaisse-Lagae, Diabetes, 18, insulin secretion in particular, has found ample experimental 365 (1970). support (22-26). 23. Matthews, E. K., The Structure and Metabolism of Pan- The microtubular and the as creatic Islets, ed. S. Falkmer, B. Hellman, and I. B. Taljedal system process of emiocytosis (Pergamon Press, Oxford, 1970), p. 163. described by Lacy (27) (IV) or a form of microvesicular secre- 24. Douglas, W. W., Brit. J. Pharnacol., 34, 451 (1968). tion as proposed by Orci (28) (not included in the scheme) are 25. Milner, D. G., and C. N. Hales, Diabetotologia, 3, 47 involved in the Ca++-dependent hormone transport and re- (1967). lease. The endocrine response can be elicited and can almost 26. Grodsky, G. M., and L. L. Bennett, Diabetes, 15, 910 certainly be modified by the adenyl cyclase system, as indi- (1966). 27. Lacy, P. E., S. L. Howell, D. A. Young, and C. J. Fink, cated by numerous reports (7-9, 12, 17, 29-31). 3':5'-Cyclic Nature (London), 219, 1177 (1968). AMP formed in the process is released from the ,B-cells (in 28. Orci, L., W. Stauffacher, D. Beaven, A. E. Lambert, analogy to events found in the liver (32)) or can increase the A. E. Renold, and C. Rouiller, Acta Diabetol. Latina, 6, Suppl. 1, intracellular pool of the nucleotide. 271 (1969). Facilitated diffusion of glucose into the cell is extremely 29. Cerasi, E., and R. Luft, Horm. Metab. Res., 1, 162 (1969). 30. Cerasi, E., S. Effendic, and R. Luft, Lancet, 301 (1969). fast, and phosphorylation is rate limiting (11, 20) for further 31. Porte, D., Jr., Diabetes, 15, 543 (1966). metabolism (II). Metabolism (i.e., alterations of the inter- 32. Hardman, J. G., J. W. Davis, and E. W. Sutherland, mediate and/or cofactor pattern) influences all or selected J. Biol. Chem., 244, 6354 (1969). components of the complex system by positive or negative 33. Morris, G. E., and A. Korner, Biochim. Biophys. Acta, 208, 404 (1970). feedback processes. Glucose itself, or factors related to its 34. Taylor, K. W., in "Etiology of Diabetes Mellitus and Its metabolism, regulate de novo synthesis of insulin (not included Complications," Ciba, Foundation Colliquia on Endocrinology XV in the scheme) (33, 34). (1964), p. 89. Downloaded by guest on September 29, 2021